The invention relates to methods for determining the pulmonary surfactant protein SP-C, to SP-C-specific antibodies and to the provision of a reagent kit for carrying out the methods.
The lungs of all vertebrates contain a substance mixture called xe2x80x9cpulmonary surfactantxe2x80x9d. It has surface-active properties and reduces surface tension in the alveolar region of the lungs. In addition to phos-pholipids such as dipalmitoylphosphatidylcholine (DPPC) and phosphatidylglycerol (PG), the pulmonary surfactant contains proteins as further essential components. To date, four different surfactant proteins have been described, which are designated SP-A, SP-B, SP-C and SP-D, corresponding to the order in which they were discovered (Possmayer, F., A Proposed Nomenclature for Pulmonary Surfactant-associated Proteins. Am. Rev. Respir. Dis. 1988, 138, 990-998). The term SP stands for surfactant protein (surfactant-associated protein, SP).
SP-B and in particular SP-C are strongly hydrophobic proteins. The total proportion of hydrophobic proteins in the pulmonary surfactant is approximately 1% (Curstedt, T., Jxc3x6rnvall, H., Robertson, B., Bergmann, T., Bergren, P.: Two Hydrophobic Low-Molecular-Mass Protein Fractions of Pulmonary Surfactant. Characterization and Biophysical Activity. Eur. J. Biochem. 1987, 168, 255-262).
The determination (detection and in particular quantification) of these hydrophobic proteins in samples of pulmonary surfactant (for example from lung lavage) frequently gives unsatisfactory results when the techniques employed for hydrophilic proteins are used, if it is possible at all. The methods which are customarily used for separating and determining hydrophilic proteins, such as, for example, xe2x80x9cWestern blottingxe2x80x9d or ELISA (Enzyme-Linked Immunosorbent Assay) techniques can be applied to hydrophobic proteins only under certain conditions, since on the one hand the xe2x80x9cWestern blotxe2x80x9d per se is semiquantitative and, on the other hand, the use of an ELISA is very problematic, since it can generally only be carried out with proteins which are soluble in aqueous systems. In most cases, only traces of the analytes to be quantified are present. Furthermore, in the samples to be analyzed, SP-B and SP-C are frequently associated with other hydrophobic substances (for example lipids), which renders quantification by customary methods even more difficult. In the ELISA, the substance to be examined is usually not separated prior to the determination from the other components which are contained in the mixture. Van Eijk and co-workers (Van Eijk, M., De Haas, C. G. M. and Haagsman, H. P.: Quantitative Analysis of Pulmonary Surfactant Proteins B and C. Analytical Biochemistry 1995, 232, 231-237) describe a process for quantifying SP-C and SP-B in samples of pulmonary surfactant. Extracts containing surfactant proteins are separated by high-pressure liquid chromatography, and SP-B or SP-C are detected and quantified by absorption at 228 nm. (The detection limits are at 1 xcexcg of SP-B and 4 xcexcg of SP-C.)
For SP-B, a quantitative detection by ELISA technique is described (Krxc3xa4mer, H. J., Schmidt, R., Gxc3xcnther, A., Becher, G., Suzuki, Y., Seeger, W.: ELISA Technique for Quantification of Surfactant Protein B (SP-B) in Bronchoalveolar Lavage Fluid. Am. J. Respir. Crit. Care Med. 1995, 152, 1540-1544). An immunoassay for the determination of SP-C has hitherto not been described, apparently because the preparation of SP-C-specific antibodies has not been successful (Beers, M. F., Wali, A., Eckenhoff, M. F., Feinstein, S. I., Fisher, J. H. and Fisher, A. B.: Am. J. Respir. Cell Mol. Biol. 1992, 7, 368-378).
It is an object of the invention to provide processes permitting the determination of pulmonary surfactant protein SP-C in a sample in a simple manner and with high sensitivity.
Surprisingly, we have succeeded in providing antibodies which are specific for SP-C, and thus allow SP-C to be determined by an immunological method.
Accordingly, the invention provides processes for determining SP-C in a sample, where the determination is carried out by an immunological method.
Further subject matters follow from the subclaims.
In the context of the invention, the determination of SP-C is to be understood as the detection and, in particular, the quantification of SP-C.
In the context of the invention, the term xe2x80x9cSP-Cxe2x80x9d is to be understood, in analogy to the nomenclature proposed by Possmayer (Possmayer, F.: A Proposed Nomenclature for Pulmonary Surfactant-associated Proteins. Am. Rev. Respir. Dis. 1988, 138, 990-998), as the xe2x80x9cfamilyxe2x80x9d of surfactant proteins which is present in natural pulmonary surfactant or the amnionic fluid of mammals designated SP-C.
Furthermore, the term xe2x80x9cSP-Cxe2x80x9d also includes chemically synthesized or recombinantly prepared SP-C and modifications of SP-C, for example those modifications where one or more amino acids are missing or have been replaced by other amino acids. Chemically synthesized or recombinantly prepared SP-C and modifications of SP-C are described, for example, in WO91/18015, WO91/00871, WO89/04326, WO93/21225 and also in WO95/32992.
SP-C is preferably understood as meaning the surfactant protein SP-C which is present in human pulmonary surfactant or in human amnionic fluid.
In the context of the present invention, xe2x80x9cimmunological methodsxe2x80x9d are understood as meaning analytical methods based on immunochemistry, in particular on an antigen-antibody reaction. Examples of immunological methods include immunoassays such as radioimmunoassay (RIA), enzyme immunoassay (EIA, combined with solid-phase technique: ELISA) or else immunofluorescence assays. The immunoassay is carried out by exposing the sample to be investigated to an SP-C-binding antibody and detecting and quantifying the amount of antibody which binds to SP-C. In these assays, detection and quantification is carried out directly or indirectly in a known manner. Thus, detection and quantification of the antigen-antibody complexes is made possible by using suitable labels which may be carried by the antibody directed against SP-C and/or by a secondary antibody directed against the primary antibody. Depending on the type of the abovementioned immunoassays, the labels are, for example, radioactive labels, fluorescent dyes or else enzymes, such as phosphatase or peroxidase, which can be detected and quantified with the aid of a suitable substrate.
In one embodiment of the invention, the immunological method is carried out with the aid of a suitable solid phase. Suitable solid phases which may be mentioned include the customary commercial microtiter plates made of polystyrene or membranes (for example made of polyvinylidene difluoride, PVDF) which are customarily used for the ELISA technique. Surprisingly, it has been found that even chromatography plates are suitable for use as solid phase in the process according to the invention. The implementation of the process according to the invention using chromatography plates is hereinbelow also referred to as immuno-TLC.
To carry out the process according to the invention, the sample is applied to the solid phase. The sample is preferably a solution of SP-C in a suitable solvent, and the solvent is evaporated after the sample has been applied to the solid phase. To prepare a solution of the sample to be investigated in a suitable solvent, it is advantageous to use organic solvents or solvent mixtures which have proved to be suitable for solubilizing hydrophobic proteins. Examples which may be mentioned are short-chain alcohols, in particular methanol, ethanol, 2-propanol (isopropanol) or n-propanol. Furthermore, in connection with immuno-TLC, mixtures of non-polar and polar solvents were found to be useful, suitable non-polar solvents being, in particular, chloroform, methylene chloride and toluene, and suitable polar solvents being short-chain alcohols. Chloroform/methanol mixtures may be mentioned as being particularly preferred.
If desired, small amounts of water and/or acid or base may be added to the abovementioned solvents or solvent mixtures to improve the solvent properties.
If the SP-C content of a bronchoalveolar lavage (BAL) sample or a sample of amnionic fluid is to be determined with the aid of immuno-TLC, it is advantageous to transfer the SP-C prior to application to the solid phase from the aqueous phase (BAL sample, amnionic fluid) to a suitable organic solvent or solvent mixture. This is expediently carried out by extraction with suitable organic solvents or solvent mixtures, for example by the extraction method of Bligh-Dyer (Can. J. Biochem. Physiol. 1959, 37, 911-917) and subsequent Folch washing (J. Biol. Chem. 1957, 226, 497-509).
If the SP-C content of a bronchoalveolar lavage (BAL) sample or a sample of amnionic fluid is to be determined with the aid of the ELISA technique, it is advantageous to dilute the sample with a suitable water-miscible solvent or solvent mixture. Suitable water-miscible solvents or solvent mixtures which may be mentioned are, in particular, short-chain alcohols, such as ethanol and isopropanol, or mixtures thereof.
Moreover, it has been found to be expedient for the method according to the invention to separate or remove interfering components in the sample to be investigated on the solid phase prior to the immunological detection of SP-C in the sample. This is particularly advantageous in cases where in the samples to be investigated other components, such as other surfactant proteins or else lipids, are present in addition to SP-C.
If chromatography plates are used as the solid phase in the method according to the invention, the sample can be separated by thin-layer chromatography. To this end, the sample is, after application and evaporation of the solvent, subjected to thin-layer chromatography. Suitable chromatography plates are all plates whose coating is suitable for separating hydrophobic mixtures in organic media. The HPTLC plates sold by Merck Darmstadt under the trade name Diol, which have a modified silica matrix, have been found to be particularly suitable in this context. Suitable solvents for the thin-layer chromatography of hydrophobic proteins are organic solvents and solvent mixtures. Particularly expedient is the use of mixtures of non-polar and polar solvents, suitable non-polar solvents being, in particular, chloroform, methylene chloride and toluene, and suitable polar solvents being short-chain alcohols, in particular methanol, ethanol and isopropanol. If desired, it is possible to add small amounts of water and/or acid or base. Preference is given to mixtures of chloroform and methanol to which small amounts of water and ammonia are added. The application of the samples to the plates and the practice of the separation are carried out in a customary manner, for example by means of customary commercial apparatuses. The sample to be applied is preferably a solution of SP-C in an organic solvent. In preparation for the immunological detection, the chromatography plates are dried after the separation by thin-layer chromatography.
It has surprisingly been found that, if the solid phase used is a polystyrene microtiter plate customarily used in the ELISA technique, interfering components in the sample can be removed after the application of the samples by one or more selective washing steps. To this end, BAL samples to be investigated are expediently diluted prior to application with a water-soluble organic solvent (for example an alcohol such as isopropanol) and adjusted to a pH below 7 (preferably pH 3-4). If desired, variable adsorption of the SP-C on the surface of the plate caused by inhomogeneities of the biological samples can be minimized. This can be effected, for example, after the application and the drying of the samplexe2x80x94and before a washingxe2x80x94by re-dissolution in a suitable solvent, such as, for example, trifluoroethanol, followed by a drying step. Interfering components can be removed, after application of the samples and evaporation of the solvent, by washing with suitable solvents. Thus, interfering lipids, for example, can be removed by one or more washings with methanol.
In preparation for the immunological detection, the solid phase, if desired, is pretreated in a suitable manner. Unspecific binding sites on the solid phase, for example, can be saturated using a suitable blocking solution, such as gelatin, or a protein solution. The solid phase is subsequently incubated with a solution of the SP-C-specific antibody. If this antibody is not labeled, detection and quantification can be carried out with the aid of a labeled secondary antibody which recognizes the primary antibody. To this end, excess primary antibody is removed by washing and the plate is then incubated with a labeled secondary antibody. After removal of excess antibody by washing, detection and quantification of the antigen-antibody complexes is carried out with the aid of the label.
Labels which are suitable for the detection and quantification step and which can be carried by the primary or secondary antibody are known to the person skilled in the art. Examples which may be mentioned include radioactive labels, fluorescent dyes and, preferably, enzymes such as phosphatase or peroxidase, which permit calorimetric or chemoluminescent detection. Depending on the label used, the subsequent detection or quantification is carried out in a known manner.
When carrying out the method according to the invention with the aid of the ELISA technique, the antigen-antibody complexes are preferably detected and quantified via an enzyme-catalyzed color reaction using a peroxidase-conjugated antibody and ABTS (2,2xe2x80x2-azino-di[3-ethylbenzothiazolinesulfonate]) as substrate. However, it is also possible to use other chromogenic, chemoluminescent or fluorogenic methods known to the person skilled in the art for the detection.
When carrying out the method according to the invention with the aid of immuno-TLC, the antigen-antibody complexes are preferably detected and quantified via an enzyme-catalyzed chemoluminescence reaction using Luminol(copyright) as substrate. However, it is also possible to use other chromogenic, chemoluminescent or fluorogenic methods known to the person skilled in the art for the detection.
As already mentioned, we have surprisingly succeeded in providing antibodies which are specific for SP-C, in particular for the SP-C which is present in human pulmonary surfactant or amnionic fluid. The invention therefore also provides an antibody which is specific for SP-C, in particular specific for SP-C which is present in human pulmonary surfactant or amnionic fluid. The antibody is furthermore advantageously characterized by the fact that it does not show any cross reactivity with other surfactant proteins such as SP-A and SP-B.
The SP-C-specific antibodies are preferably polyclonal antibodies.
The SP-C-specific antibodies are prepared analogously to processes known to the person skilled in the art (as described, for example, in Antibodies: A Laboratory Manual. Eds. E. Hariow and D. Lane. Cold Spring Harbor Laboratory 1987).
The preparation of polyconal antibodies directed against SP-C is expediently carried out by known immunization methods. Here, it has been found to be advantageous to use recombinant SP-C (herein-below also referred to as rSP-C) as antigen in the immunization process, and to employ larger amounts of antigen than are usually used, and to increase the number of antigen administrations. In the immunization process, between 0.5 mg and 2 mg (preferably 1 mg) of rSP-C are expediently employed per administration of antigen, where the number of antigen administrations is advantageously between 5 and 7, preferably at an interval of from three to six weeks (preferably four weeks). Furthermore, it has been found to be advantageous to use the rSP-C in the form of insoluble particles (aggregates or precipitates), or coupled to a carrier molecule (for example protein or beads). Aggregates and precipitates can be obtained in a customary manner, expediently as described in the xe2x80x9cExamplesxe2x80x9d section.
Suitable rSP-C for use in the immunization process which may be mentioned includes recombinant human-identical dipalmitoylated SP-C (rhSP-C2Pam) or recombinant modifications of SP-C. Recombinant modifications of SP-C which may be mentioned include, for example, non-palmitoylated human-identical SP-C (rhSP-C) or the SP-C modification with 34 amino acids described in the international patent application WO95/32992, where the amino acids in positions 4 and 5 of the amino acid sequence are phenylalanine and the amino acid in position 32 of the amino acid sequence is isoleucine (the stated positions within the amino acid sequence are based on the amino acid sequence for the peptides of the formula I described in WO95132992). The preparation of this peptide, which is designated rSP-C34 (FF/1), by genetic engineering is likewise described in WO95/32992.
The invention therefore also relates to a process for preparing polyclonal antibodies directed against SP-C by immunization, in particular against human SP-C, where the antigen component used for the immunization is recombinant SP-C. By way of example, the preparation of antibodies which are specific for SP-C, and an immunological determination of SP-C in a sample, are described below.